WO2020157694A1 - Antiviral nucleosides and derivatives thereof - Google Patents

Antiviral nucleosides and derivatives thereof Download PDF

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Publication number
WO2020157694A1
WO2020157694A1 PCT/IB2020/050747 IB2020050747W WO2020157694A1 WO 2020157694 A1 WO2020157694 A1 WO 2020157694A1 IB 2020050747 W IB2020050747 W IB 2020050747W WO 2020157694 A1 WO2020157694 A1 WO 2020157694A1
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Prior art keywords
compound
formula
pharmaceutically acceptable
benzyloxy
compounds
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PCT/IB2020/050747
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English (en)
French (fr)
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WO2020157694A9 (en
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Leonid Beigelman
Guangyi Wang
Natalia Dyatkina
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Janssen Biopharma, Inc.
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Priority to MX2021009278A priority Critical patent/MX2021009278A/es
Priority to CN202080012024.2A priority patent/CN113382994A/zh
Priority to EP20704597.2A priority patent/EP3917922A1/en
Priority to JP2021544345A priority patent/JP2022518943A/ja
Priority to CA3128455A priority patent/CA3128455A1/en
Priority to AU2020215175A priority patent/AU2020215175A1/en
Priority to KR1020217026477A priority patent/KR20210121105A/ko
Priority to US17/426,974 priority patent/US20230365608A1/en
Publication of WO2020157694A1 publication Critical patent/WO2020157694A1/en
Publication of WO2020157694A9 publication Critical patent/WO2020157694A9/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H7/00—Compounds containing non-saccharide radicals linked to saccharide radicals by a carbon-to-carbon bond
    • C07H7/06—Heterocyclic radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/24—Heterocyclic radicals containing oxygen or sulfur as ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/661—Phosphorus acids or esters thereof not having P—C bonds, e.g. fosfosal, dichlorvos, malathion or mevinphos
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/661—Phosphorus acids or esters thereof not having P—C bonds, e.g. fosfosal, dichlorvos, malathion or mevinphos
    • A61K31/6615—Compounds having two or more esterified phosphorus acid groups, e.g. inositol triphosphate, phytic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/7056—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing five-membered rings with nitrogen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
    • C07F9/65586—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system at least one of the hetero rings does not contain nitrogen as ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H23/00—Compounds containing boron, silicon, or a metal, e.g. chelates, vitamin B12
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/582—Recycling of unreacted starting or intermediate materials

Definitions

  • the present invention relates to nucleoside compounds and derivatives thereof, pharmaceutical compositions comprising these compounds, processes for preparing same and their use in treating an orthomyxovirus or influenza infection in animals, particularly humans.
  • the viruses of the Orthomyxoviridae family are negative-sense, single-stranded RNA viruses.
  • the Orthomyxoviridae family contains several genera including Influenza virus A, Influenza virus B, Influenza virus C, Isavirus and Thogotovirus.
  • Influenza viruses can cause respiratory viral infections, including upper and lower respiratory tract viral infections. Respiratory viral infections are a leading cause of death of millions of people each year. Upper respiratory tract viral infections involve the nose, sinuses, pharynx and/or larynx. Lower respiratory tract viral infections involve the respiratory system below the vocal cords, including the trachea, primary bronchi and lungs.
  • Influenza is a negative sense, single stranded RNA virus and a member of the Orthomyxoviridae family. There are currently three species of influenza; influenza A, influenza B and influenza C. Influenza A has a lipid membrane derived from the host cell, which contains the hemagglutinin, neuraminidase and M2 proteins that project from the surface of the virus. Influenza A has been further classified based on two viral surface proteins, namely hemagglutinin (H or HA) and neuraminidase (N). There are approximately 16 Hantigens (H1 to H16) and 9 N antigens (N1 to N9).
  • H or HA hemagglutinin
  • N neuraminidase
  • Influenza A includes several subtypes, including H1 N1 , H1 N2, H2N2, H3NI, H3N2, H3N8, H5N1 , H5N2, H5N3, H5N8, H5N9, H7N1 , H7N2, H7N3, H7N4, H7N7, H7N9, H9N2 and H10N7.
  • the influenza virus polymerase is a heterotrimer composed of three subunits, polymerase acid (PA), polymerase basic 1 (PB1 ) and polymerase basic 2 (PB2). This polymerase is responsible for replication and transcription of the viral RNA in the nuclei of infected cells.
  • the PA subunit contains the endonuclease active site. The endonuclease activity of the PA cleaves the cellular mRNA, which is then used by the PB1 subunit as a primer for the viral mRNA synthesis.
  • Influenza viruses can be transmitted from person to person via direct contact with infected secretions and/or contaminated surfaces or objections.
  • Complications from an influenza viral infection include pneumonia, bronchitis, dehydration, and sinus and ear infections.
  • many countries have adopted vaccinations as a preventive measure with limited success due to the difficulty of predicting which influenza strain(s) will dominate in the impending flu season. Even when the correct strain(s) are identified, vaccinations offer incomplete immunity against the flu as evidenced by the spike in hospitalizations and deaths for the 2017-2018 flu season. Universal vaccines capable of protecting against all flu strains have not yet been developed despite active research in this area.
  • Medications currently approved by the FDA against influenza infections include a limited number of neuraminidase inhibitors and M2 protein inhibitors.
  • approved neuraminidase inhibitors and M2 protein inhibitors include amantadine, rimantadine, Relenza® (zanamivir,
  • HET is a heteroaryl selected from the group consisting of:
  • the compounds of Formula (I) are compounds selected from those species described or exemplified in the detailed description below.
  • the invention relates to enantiomers and diastereomers of the compounds of Formula (I) (as well as Formula (II), Formula (III), and Formula (IV)), as well as the pharmaceutically acceptable salts.
  • compositions for treating an orthomyxovirus viral infection comprising an effective amount of at least one compound selected from compounds of Formula (I) (as well as Formula (II), Formula
  • compositions according to the invention may further comprise one or more pharmaceutically acceptable excipients.
  • the invention is directed to a method of treating a subject suffering from, or diagnosed with an orthomyxovirus viral infection, comprising administering to the subject in need of such treatment an effective amount of at least one compound selected from compounds of Formula (I) (as well as Formula (II),
  • Formula (III), and Formula (IV) pharmaceutically acceptable salts of compounds of Formula (I) (as well as Formula (II), Formula (III), and Formula (IV)), pharmaceutically acceptable prodrugs of compounds of Formula (I) (as well as Formula (II), Formula (III), and Formula (IV)), and pharmaceutically active metabolites of compounds of Formula (I) (as well as Formula (II), Formula (III), and Formula (IV)). Additional embodiments of methods of treatment are set forth in the detailed description.
  • Additional embodiments of this invention include methods of making compounds of Formula (I) (as well as Formula (II), Formula (III), and Formula (IV)) ,
  • An object of the present invention is to overcome or ameliorate at least one of the disadvantages of the conventional methodologies and/or prior art, or to provide a useful alternative thereto.
  • alkyl refers to a straight or branched hydrocarbon chain that comprises a fully saturated (no double or triple bonds) hydrocarbon group.
  • the alkyl group may have 1 to 20 carbon atoms (whenever it appears herein, a numerical range such as“1 to 20” refers to each integer in the given range; e.g.,“1 to 20 carbon atoms” means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms, although the present definition also covers the occurrence of the term“alkyl” where no numerical range is designated).
  • the alkyl group may also be a medium size alkyl having 1 to 10 carbon atoms.
  • the alkyl group could also be a lower alkyl having 1 to 6 carbon atoms.
  • the alkyl group of the compounds may be designated as“C1-C6 alkyl” or similar designations.
  • “C1-C6 alkyl” indicates that there are one to six carbon atoms in the alkyl chain, i.e. , the alkyl chain is selected from methyl, ethyl, propyl, iso-propyl, n-butyl, iso butyl, sec-butyl, and t-butyl.
  • Typical alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl (straight and branched) and hexyl (straight and branched).
  • the alkyl group may be substituted or unsubstituted.
  • heteroaryl or ⁇ ET refers to a monocyclic or fused bicyclic
  • heterocycle (ring structure having ring atoms selected from carbon atoms and up to four heteroatoms selected from nitrogen, oxygen, and sulfur) having from 3 to 9 ring atoms per heterocycle.
  • heteroaryl groups include the following entities, in the form of properly bonded moieties:
  • heteroaryl groups listed or illustrated above are not exhaustive, and that additional species within the scope of these defined terms may also be selected.
  • substituted means that the specified group or moiety bears one or more substituents.
  • unsubstituted means that the specified group bears no substituents.
  • optionally substituted means that the specified group is unsubstituted or substituted by one or more substituents.
  • substitution is meant to occur at any valency-allowed position on the system. In cases where a specified moiety or group is not expressly noted as being optionally substituted or substituted with any specified substituent, it is understood that such a moiety or group is intended to be unsubstituted.
  • protecting group and“protecting groups” as used herein refer to any atom or group of atoms that is added to a molecule in order to prevent existing groups in the molecule from undergoing unwanted chemical reactions.
  • Examples of protecting group moieties are described in T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3. Ed. John Wiley & Sons, 1999, and in J.F.W. McOmie, Protective Groups in Organic Chemistry Plenum Press, 1973, both of which are hereby
  • protecting group moiety may be chosen in such a way, that they are stable to certain reaction conditions and readily removed at a convenient stage using methodology known from the art.
  • a non-limiting list of protecting groups include benzyl; substituted benzyl; alkylcarbonyls and alkoxycarbonyls (e.g., t-butoxycarbonyl (BOC), acetyl and isobutyryl); arylalkylcarbonyls and arylalkoxycarbonyls (e.g.,
  • tetrahydropyranyl ether substituted ethyl ether; a substituted benzyl ether; silyls (e.g., trimethylsilyl, triethylsilyl, triisopropylsilyl, t-butyldimethylsilyl, tri-/so-propylsilyloxymethyl, [2-(trimethylsilyl)ethoxy]methyl and t-butyldiphenylsilyl); esters (e.g. benzoate ester); carbonates (e.g. methoxymethylcarbonate); sulfonates (e.g. tosylate and mesylate); acyclic ketal (e.g.
  • cyclic ketals e.g., 1 ,3-dioxane and 1 ,3-dioxolane
  • acyclic acetal cyclic acetal; acyclic hemiacetal; cyclic hemiacetal; dithioacetals (both cyclic and acyclic); dithioketals (both cyclic and acyclic) (e.g., S,S’- dimethyl, S,S’-diethyl, S,S’-diispropyl, 1 ,3-dithiane and 1 ,3-dithiolane); orthoesters (including cyclic orthoesters, such as cyclic orthoformates); carbamates (e.g., N- phenylcarbamate) and triarylmethyl groups (e.g., trityl, monomethoxytrityl (MMTr), 4,4'- dimeth
  • leaving group refers to any atom or moiety that is capable of being displaced by another atom or moiety in a chemical reaction. More specifically, in some embodiments,“leaving group” refers to the atom or moiety that is displaced in a nucleophilic substitution reaction. In some embodiments,“leaving groups” are any atoms or moieties that are conjugate bases of strong acids. Examples of suitable leaving groups include, but are not limited to, tosylates, mesylates, trifluoroacetates and halogens (e.g., I, Br, and Cl).
  • Non-limiting characteristics and examples of leaving groups can be found, for example in Organic Chemistry, 2d ed., Francis Carey (1992), pages 328-331 ; Introduction to Organic Chemistry, 2d ed., Andrew Streitwieser and Clayton Heathcock (1981 ), pages 169-171 ; and Organic Chemistry, 5 th ed., John McMurry (2000), pages 398 and 408; all of which are incorporated herein by reference for the limited purpose of disclosing characteristics and examples of leaving groups.
  • pharmaceutically acceptable salt refers to a salt of a compound that does not cause significant irritation to an organism to which it is administered and does not abrogate the biological activity and properties of the compound.
  • the salt is an acid addition salt of the compound.
  • Pharmaceutical salts can be obtained by reacting a compound with inorganic acids such as hydrohalic acid (e.g., hydrochloric acid or hydrobromic acid), sulfuric acid, nitric acid and phosphoric acid.
  • compositions can also be obtained by reacting a compound with an organic acid such as aliphatic or aromatic carboxylic or sulfonic acids, for example formic, acetic, succinic, lactic, malic, tartaric, citric, ascorbic, nicotinic, methanesulfonic, ethanesulfonic, p-toluensulfonic, salicylic or naphthalenesulfonic acid.
  • organic acid such as aliphatic or aromatic carboxylic or sulfonic acids
  • Pharmaceutical salts can also be obtained by reacting a compound with a base to form a salt such as an ammonium salt, an alkali metal salt, such as a sodium or a potassium salt, an alkaline earth metal salt, such as a calcium or a magnesium salt, a salt of organic bases such as dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, C1-C7 alkylamine, cyclohexylamine, triethanolamine, ethylenediamine, and salts with amino acids such as arginine and lysine.
  • a salt such as an ammonium salt, an alkali metal salt, such as a sodium or a potassium salt, an alkaline earth metal salt, such as a calcium or a magnesium salt, a salt of organic bases such as dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, C1-C7 alkylamine, cyclohexylamine
  • the term“comprising” is to be interpreted synonymously with the phrases “having at least” or “including at least”.
  • the term “comprising” means that the process includes at least the recited steps, but may include additional steps.
  • the term “comprising” means that the compound, composition or device includes at least the recited features or components, but may also include additional features or components.
  • a group of items linked with the conjunction‘and’ should not be read as requiring that each and every one of those items be present in the grouping, but rather should be read as‘and/or’ unless expressly stated otherwise.
  • a group of items linked with the conjunction‘or’ should not be read as requiring mutual exclusivity among that group, but rather should be read as‘and/or’ unless expressly stated otherwise.
  • each center may independently be of R-configuration or S-configuration or a mixture thereof.
  • the compounds provided herein may be enantiomerically pure, enantiomerically enriched, racemic mixture, diastereomerically pure, diastereomerically enriched, or a stereoisomeric mixture.
  • each double bond may independently be E or Z a mixture thereof.
  • valencies are to be filled with hydrogens or isotopes thereof, e.g., hydrogen-1 (protium) and hydrogen-2 (deuterium).
  • each chemical element as represented in a compound structure may include any isotope of said element.
  • a hydrogen atom may be explicitly disclosed or understood to be present in the compound.
  • the hydrogen atom can be any isotope of hydrogen, including but not limited to hydrogen-1 (protium) and hydrogen-2 (deuterium).
  • reference herein to a compound encompasses all potential isotopic forms unless the context clearly dictates otherwise.
  • the methods and combinations described herein include crystalline forms (also known as polymorphs, which include the different crystal packing arrangements of the same elemental composition of a compound), amorphous phases, salts, solvates, and hydrates.
  • the compounds described herein exist in solvated forms with pharmaceutically acceptable solvents such as water, ethanol, or the like.
  • the compounds described herein exist in unsolvated form.
  • Solvates contain either stoichiometric or non-stoichiometric amounts of a solvent, and may be formed during the process of crystallization with pharmaceutically acceptable solvents such as water, ethanol, or the like. Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is alcohol.
  • the compounds provided herein can exist in unsolvated as well as solvated forms. In general, the solvated forms are considered equivalent to the unsolvated forms for the purposes of the compounds and methods provided herein.
  • HET is a heteroaryl selected from the group consisting of:
  • An additional embodiment of the invention is a compound of Formula (I) wherein al embodiment of the invention is a compound of Formula (I) wherein
  • An additional embodiment of the invention is a compound of Formula (I) wherein S-N
  • HET is A - .
  • An additional embodiment of the invention is a compound of Formula (II), and pharmaceutically acceptable salts, stereoisomers, isotopic variants, N-oxides, or solvates thereof,
  • An additional embodiment of the invention is a compound of Formula (III), and pharmaceutically acceptable salts, stereoisomers, isotopic variants, N-oxides, or solvates thereof,
  • R 7 is H or two R 7 members come together to form a 5-membered ring substituted with OCH3;
  • a further embodiment of the current invention is a compound selected from the group consisting of:
  • An additional embodiment of the invention is a compound of Formula (IV), and pharmaceutically acceptable salts, stereoisomers, isotopic variants, N-oxides, or solvates thereof,
  • HET is a heteroaryl selected from the group consisting of:
  • a further embodiment of the current invention is a compound as shown below in
  • compositions that can include an effective amount of one or more compounds described herein (e.g., a compound of Formula (I) (as well as Formulas (II), (III) and (IV)), or a pharmaceutically acceptable salt thereof) and a pharmaceutically acceptable carrier, diluent, excipient or combination thereof.
  • compositions can also be obtained by reacting compounds with inorganic or organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, and salicylic acid.
  • inorganic or organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, and salicylic acid.
  • Pharmaceutical compositions will generally be tailored to the specific intended route of administration.
  • physiologically acceptable defines a carrier, diluent or excipient that does not abrogate the biological activity and properties of the compound.
  • a“carrier” refers to a compound that facilitates the incorporation of a compound into cells or tissues.
  • DMSO dimethyl sulfoxide
  • a“carrier” refers to a compound that facilitates the incorporation of a compound into cells or tissues.
  • DMSO dimethyl sulfoxide
  • a“diluent” refers to an ingredient in a pharmaceutical composition that lacks pharmacological activity but may be pharmaceutically necessary or desirable.
  • a diluent may be used to increase the bulk of a potent drug whose mass is too small for manufacture and/or administration. It may also be a liquid for the dissolution of a drug to be administered by injection, ingestion or inhalation.
  • a common form of diluent in the art is a buffered aqueous solution such as, without limitation, phosphate buffered saline that mimics the composition of human blood.
  • an“excipient” refers to an inert substance that is added to a pharmaceutical composition to provide, without limitation, bulk, consistency, stability, binding ability, lubrication, disintegrating ability etc., to the composition. Suitable excipients can be found in the Handbook of Pharmaceutical Excipients, published by the American Pharmaceutical Association, which is incorporated herein by reference. A “diluent” is a type of excipient.
  • compositions described herein can be administered to a human patient perse, or in pharmaceutical compositions where they are mixed with other active ingredients, as in combination therapy, or carriers, diluents, excipients or combinations thereof. Proper formulation is dependent upon the route of administration chosen. Techniques for formulation and administration of the compounds described herein are known to those skilled in the art.
  • compositions disclosed herein may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or tableting processes. Additionally, the active ingredients are contained in an amount effective to achieve its intended purpose. Many of the compounds used in the pharmaceutical combinations disclosed herein may be provided as salts with pharmaceutically compatible counterions.
  • an effective amount of one or more compounds of Formula (I), or a pharmaceutically acceptable salt thereof, and/or a pharmaceutical composition that includes one or more compounds described herein (e.g., a compound of Formula (I), or a pharmaceutically acceptable salt thereof) can be administering intramuscular.
  • an effective amount of one or more compounds of Formula (I), or a pharmaceutically acceptable salt thereof, and/or a pharmaceutical composition that includes one or more compounds described herein can be administering intranasal.
  • an effective amount of one or more compounds of Formula (I), or a pharmaceutically acceptable salt thereof, and/or a pharmaceutical composition that includes one or more compounds described herein can be administering intradermal.
  • an effective amount of one or more compounds of Formula (I), or a pharmaceutically acceptable salt thereof, and/or a pharmaceutical composition that includes one or more compounds described herein (e.g., a compound of Formula (I), or a pharmaceutically acceptable salt thereof) can be administering orally.
  • one or more compounds described herein can be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated.
  • injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • Pharmaceutical compositions for intranasal delivery may also include drops and sprays often prepared to assist in simulating nasal secretions.
  • compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may for example comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser may also be accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration. Such notice, for example, may be the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert.
  • Compositions that can include a compound described herein formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
  • An additional embodiment of the invention is a pharmaceutical composition comprising and effective amount of at least one compound in Table 1 , as well as pharmaceutically acceptable salts, N-oxides or solvates of compounds of Table 1 , pharmaceutically acceptable prodrugs of compounds of Table 1 , and pharmaceutically active metabolites of Table 1 ; and at least one pharmaceutically acceptable excipient.
  • An additional embodiment of the invention is a pharmaceutical composition comprising and effective amount of at least one compound in Table 2, as well as pharmaceutically acceptable salts, N-oxides or solvates of compounds of Table 2, pharmaceutically acceptable prodrugs of compounds of Table 2, and pharmaceutically active metabolites of Table 2; and at least one pharmaceutically acceptable excipient.
  • enantiomers and diastereomers of the compounds of Formula (I) are also within the scope of the invention.
  • pharmaceutically acceptable salts, N-oxides or solvates of the compounds of Formula (I) are also within the scope of the invention.
  • pharmaceutically acceptable prodrugs of compounds of Formula (I) are also within the scope of the invention, and pharmaceutically active metabolites of the compounds of Formula (I) (as well as Formulas (II), (III), and (IV)).
  • Compounds of the present invention can be provided in the form of a prodrug, namely a compound which is metabolized in vivo to the active metabolite. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in "Design of Prodrugs", ed.
  • Some embodiments described herein relate to a method of ameliorating and/or treating an orthomyxovirus infection, e.g. influenza virus infection, which can include administering an effective amount of one or more compounds described herein, or a pharmaceutical composition that includes one or more compounds described herein (e.g. , a compound of Formula (I), or a pharmaceutically acceptable salt thereof).
  • an orthomyxovirus infection e.g. influenza virus infection
  • a pharmaceutical composition that includes one or more compounds described herein (e.g. , a compound of Formula (I), or a pharmaceutically acceptable salt thereof).
  • inventions described herein relate to a method of inhibiting an orthomyxovirus viral replication, e.g. influenza viral replication, which can include contacting a cell infected with the orthomyxovirus virus, e.g. influenza viral replication, with an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof and/or a pharmaceutical composition that includes one or more compounds described herein (e.g., a compound of Formula (I), or a pharmaceutically acceptable salt thereof).
  • an effective amount of one or more compounds of Formula (I), or a pharmaceutically acceptable salt thereof, and/or a pharmaceutical composition that includes one or more compounds described herein can be used to treat and/or ameliorate an influenza viral infection.
  • an effective amount of one or more compounds of Formula (I), or a pharmaceutically acceptable salt thereof and/or a pharmaceutical composition that includes one or more compounds described herein can be used to treat and/or ameliorate an influenza viral infection.
  • an effective amount of one or more compounds of Formula (I), or a pharmaceutically acceptable salt thereof and/or a pharmaceutical composition that includes one or more compounds described herein (e.g., a compound of Formula (I), or a pharmaceutically acceptable salt thereof) can be used to
  • an effective amount of one or more compounds of Formula (I), or a pharmaceutically acceptable salt thereof, and/or a pharmaceutical composition that includes one or more compounds described herein (e.g., a compound of Formula (I), or a pharmaceutically acceptable salt thereof) can be used to inhibit the replication an influenza virus.
  • an effective amount of one or more compounds of Formula (I), or a pharmaceutically acceptable salt thereof and/or a pharmaceutical composition that includes one or more compounds described herein (e.g., a compound of Formula (I), or a pharmaceutically acceptable salt thereof) can be used to inhibit activity of the influenza polymerase complex.
  • an effective amount of one or more compounds of Formula (I), or a pharmaceutically acceptable salt thereof and/or a pharmaceutical composition that includes one or more compounds described herein can be used for inhibiting and/or reducing the endonuclease activity of an influenza endonuclease that can include contacting the active site of the endonuclease with a compound of Formula (I), or a pharmaceutically acceptable salt thereof.
  • one or more compounds described herein inhibits and/or reduces the ability of the endonuclease to cleave the mRNA.
  • influenza viral infection can be an influenza A viral infection. In other embodiments, including those embodiments in the previous paragraphs, the influenza viral infection can be an influenza B viral infection. In still other embodiments, including those embodiments in the previous paragraphs, the influenza viral infection can be an influenza C viral infection.
  • a compound of Formula (I), or a pharmaceutically acceptable salt thereof can be used to treat and/or ameliorate one or more subtypes of influenza. For example, a compound of Formula (I), or a pharmaceutically acceptable salt thereof, can be used to treat H1 N1 and/or H3N2.
  • a compound of Formula (I), or a pharmaceutically acceptable salt thereof can be used to treat H2N2, H5N1 and/or H7N9.
  • a compound described herein (a compound of Formula (I), or a pharmaceutically acceptable salt thereof) can be effective against more than 1 subtype of influenza.
  • a compound described herein (a compound of Formula (I), or a pharmaceutically acceptable salt thereof can be effective against 2, 3, 4, and/or 5 or more subtypes of influenza.
  • an effective amount of one or more compounds of Formula (I), or a pharmaceutically acceptable salt thereof, and/or a pharmaceutical composition that includes one or more compounds described herein (e.g., a compound of Formula (I), or a pharmaceutically acceptable salt thereof) can be used treat and/or ameliorate an upper respiratory viral infection attributed to (directly and/or indirectly) an influenza virus infection.
  • an effective amount of one or more compounds of Formula (I), or a pharmaceutically acceptable salt thereof, and/or a pharmaceutical composition that includes one or more compounds described herein can be used treat and/or ameliorate a lower respiratory viral infection attributed to (directly and/or indirectly) an influenza virus infection.
  • an effective amount of one or more compounds of Formula (I), or a pharmaceutically acceptable salt thereof, and/or a pharmaceutical composition that includes one or more compounds described herein can be used treat and/or ameliorate one or more symptoms of an influenza virus infection (such as those described herein).
  • an effective amount of one or more compounds of Formula (I), or a pharmaceutically acceptable salt thereof, and/or a pharmaceutical composition that includes one or more compounds described herein (e.g., a compound of Formula (I), or a pharmaceutically acceptable salt thereof) can be used treat and/or ameliorate bronchiolitis and/or tracheobronchitis due to an influenza virus infection.
  • an effective amount of one or more compounds of Formula (I), or a pharmaceutically acceptable salt thereof, and/or a pharmaceutical composition that includes one or more compounds described herein (e.g., a compound of Formula (I), or a pharmaceutically acceptable salt thereof) can be used treat and/or ameliorate pneumonia due to an influenza virus infection.
  • an effective amount of one or more compounds of Formula (I), or a pharmaceutically acceptable salt thereof, and/or a pharmaceutical composition that includes one or more compounds described herein (e.g., a compound of Formula (I), or a pharmaceutically acceptable salt thereof) can be used treat and/or ameliorate coup due to an influenza virus infection.
  • an effective amount of one or more compounds of Formula (I), or a pharmaceutically acceptable salt thereof, and/or a pharmaceutical composition that includes one or more compounds described herein (e.g., a compound of Formula (I), or a pharmaceutically acceptable salt thereof) can be used to lessen the severity of one or more symptoms of an influenza infection; examples of symptoms include, but are not limited to: fever, chills, cough, sore throat, runny nose, stuffy nose, muscle aches, body aches, headache, fatigue, vomiting and/or diarrhea.
  • the terms “treat,” “treating,” “treatment,” “therapeutic,” and “therapy” do not necessarily mean total cure or abolition of the disease or condition. Any alleviation of any undesired signs or symptoms of a disease or condition, to any extent can be considered treatment and/or therapy.
  • a therapeutically effective amount of compound can be the amount needed to alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. This response may occur in a tissue, system, animal or human and includes alleviation of the signs or symptoms of the disease being treated. Determination of an effective amount is well within the capability of those skilled in the art, in view of the disclosure provided herein.
  • the therapeutically effective amount of the compounds disclosed herein required as a dose will depend on the route of administration, the type of animal, including human, being treated, and the condition or physical characteristics of the specific animal under consideration. The dose can be tailored to achieve a desired effect, but will depend on such factors as age, weight, diet, concurrent medication and other factors which those skilled in the medical arts will recognize.
  • the term "subject” refers to an animal, preferably a mammal, most preferably a human, who has been the object of treatment or observation.“Mammal” includes, without limitation, mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cows, horses, primates, such as monkeys, chimpanzees, and apes, and humans. In some embodiments, the subject is human.
  • Suitable indicators include, but are not limited to, a reduction in viral load, a reduction in viral replication, a reduction in time to seroconversion (virus undetectable in patient serum), a reduction of morbidity or mortality in clinical outcomes, ameliorate symptoms of the disease, and/or other indicator of disease response.
  • a compound of Formula (I), or a pharmaceutically acceptable salt of the foregoing can result in one or more improvement in quality of life, such as reduced illness duration, reduced illness severity, reduced time to return to normal health and normal activity, and reduced time to alleviation of one or more symptoms of orthomyxovirus infection, compared to a subject who is untreated.
  • a compound of Formula (I), or a pharmaceutically acceptable salt of the foregoing can result in one or more improvement in quality of life, such as reduced illness duration, reduced illness severity, reduced time to return to normal health and normal activity, and reduced time to alleviation of one or more symptoms of orthomyxovirus infection, compared to a subject receiving conventional standard of care for treating influenza.
  • a compound of Formula (I), or a pharmaceutically acceptable salt of the foregoing can result in a reduced length and/or severity of one or more symptoms associated with an orthomyxovirus infection compared to an untreated subject.
  • Symptoms of an orthomyxovirus infection are described herein and include but not limited to chills, cough, myalgia (muscle pain), nasal obstruction, sore throat, fatigue, headache and fever.
  • a compound of Formula (I), or a pharmaceutically acceptable salt of the thereof can result in a reduction in one or more secondary complications associated with an orthomyxovirus infection, including but not limited to otitis media (ear inflammation), sinusitis, bronchitis and pneumonia compared to an untreated subject.
  • a compound of Formula (I), or a pharmaceutically acceptable salt of the foregoing can result in at least a 1 , 2, 3, 4, 5, 10, 15, 20, 25, 50, 75, 100-fold or more reduction in the replication of an orthomyxovirus relative to pre treatment levels in a subject, as determined after initiation of the treatment regime (for example, 10 days after initiation of treatment).
  • a compound of Formula (I), or a pharmaceutically acceptable salt of the foregoing can result in a reduction of the replication of an orthomyxovirus relative to pre-treatment levels in the range of about 2 to about 5 fold, about 10 to about 20 fold, about 15 to about 40 fold, or about 50 to about 100 fold.
  • a compound of Formula (I), or a pharmaceutically acceptable salt thereof can result in a reduction of orthomyxovirus replication in the range of 1 to 1.5 log, 1.5 log to 2 log, 2 log to 2.5 log, 2.5 to 3 log, or 3 to 3.5 log reduction of orthomyxovirus replication compared to the reduction of orthomyxovirus reduction achieved by oseltamivir (Tamiflu®), or may achieve the same reduction as that of oseltamivir (Tamiflu®) therapy in a shorter period of time, for example, in one day, two days, three days, or four days as compared to the reduction achieved after 5 days of oseltamivir (Tamiflu®) therapy.
  • influenza A strains have developed resistance to the class of influenza drugs known as adamantanes, which include amantadine and rimantadine.
  • adamantanes which include amantadine and rimantadine.
  • H1 N1 influenza viruses are known to possess resistance to oseltamivir.
  • resistance refers to a viral strain displaying a delayed, lessened and/or null response to a therapeutic agent(s).
  • a compound of Formula (I) (as well as Formulas (II), (III) and (IV)), or a pharmaceutically acceptable salt thereof, can be administered to a subject infected with a strain of influenza virus that is resistant to one or more different anti-influenza agents (for example, amantadine, rimantadine and/or oseltamivir).
  • a compound of Formula (I), or a pharmaceutically acceptable salt thereof can be administered to a subject infected with an influenza virus that is resistant to a M2 protein inhibitor.
  • a compound of Formula (I), or a pharmaceutically acceptable salt thereof can decrease the percentage of subjects that experience complications from an influenza viral infection compared to the percentage of subjects that experience complication being treated with oseltamivir.
  • the percentage of subjects being treated with a compound of Formula (I), or a pharmaceutically acceptable salt thereof, that experience complications can be 10%, 25%, 40%, 50%, 60%, 70%, 80% and 90% less compared to subjects being treated with oseltamivir.
  • a compound of Formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition that includes a compound described herein can be used in combination with one or more additional agent(s).
  • a compound of Formula (I), or a pharmaceutically acceptable salt thereof can be used in combination with one or more agents currently used in a conventional standard of care for treating influenza.
  • the additional agent can be amantadine (adamantan-1 -amine, Symmetrel®), rimantadine (Flumadine®), zanamivir (Relenza®) and oseltamivir (Tamiflu®).
  • additional agents include but are not limited to a neuraminidase inhibitor, a M2 protein inhibitor, a polymerase inhibitor, a PB2 inhibitor, peramivir ((1 S,2S,3S,4R)-3-[(1 S)-1 - acetamido-2-ethylbutyl]-4-(diaminomethylideneamino)-2-hydroxycyclopentane-1 - carboxylic acid, BioCryst Pharmaceuticals), laninamivir ((4S,5R,6R)-5-acetamido-4- carbamimidamido-6-[(1 R,2R)-3-hydroxy-2-methoxypropyl]-5,6-dihydro-4H-pyran-2- carboxylic acid), favipiravir (T-705, 6-fluoro-3-hydroxy-2-pyrazinecarboxamide), laninamivir octanoate ((3R,4S)-3-aceta
  • a compound of Formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition that includes a compound described herein, can be used in combination with oseltami
  • Type 1 interferons are known to those skilled in the art. A non-limiting list of examples include: alpha-interferons, beta-interferons, delta-interferons, omega- interferons, tau-interferons, x-interferons, consensus interferons and asialo-interferons. Type 1 interferons can be pegylated.
  • Examples of specific type 1 interferons include interferon alpha 1A, interferon alpha 1 B, interferon alpha 2A, interferon alpha 2B, pegylated-interferon alpha 2a (PEGASYS, Roche), recombinant interferon alpha 2a (ROFERON, Roche), inhaled interferon alpha 2b (AERX, Aradigm), pegylated-interferon alpha 2b (ALBUFERON, Human Genome Sciences/Novartis, PEGINTRON, Schering), recombinant interferon alpha 2b (INTRON A, Schering), pegylated interferon alpha 2b (PEG-INTRON, Schering, VIRAFERONPEG, Schering), interferon beta-1 a (REBIF, Serono, Inc. and Pfizer), consensus interferon alpha (INFERGEN, Valeant Pharmaceutical).
  • a compound of Formula (I), or a pharmaceutically acceptable salt thereof can be administered with one or more additional agent(s) together in a single pharmaceutical composition.
  • a compound of Formula (I), or a pharmaceutically acceptable salt thereof can be administered with one or more additional agent(s) as two or more separate pharmaceutical compositions.
  • a compound of Formula (I), or a pharmaceutically acceptable salt thereof can be administered in one pharmaceutical composition, and at least one of the additional agents can be administered in a second pharmaceutical composition.
  • one or more of the additional agents can be in a first pharmaceutical composition that includes a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one of the other additional agent(s) can be in a second pharmaceutical composition.
  • a compound of Formula (I), or a pharmaceutically acceptable salt thereof can be administered prior to all additional agents.
  • a compound of Formula (I), or a pharmaceutically acceptable salt thereof can be administered prior to at least one additional agent.
  • a compound of Formula (I), or a pharmaceutically acceptable salt thereof can be administered concomitantly with one or more additional agent(s).
  • a compound of Formula (I), or a pharmaceutically acceptable salt thereof can be administered subsequent to the administration of at least one additional agent.
  • a compound of Formula (I), or a pharmaceutically acceptable salt thereof can be administered subsequent to the administration of all additional agents.
  • the route of administration, exact dosage and frequency of administration depends on the particular compound of formula (I) (or formula (II), (III), or (IV)) used, the mammalian species treated, the particular condition being treated, the severity of the condition being treated, the age, weight and general physical condition of the particular patient as well as other medication the individual may be taking, as is well known to those skilled in the art. Furthermore, it is evident that said effective amount may be lowered or increased depending on the response of the treated subject and/or depending on the evaluation of the physician prescribing the compounds of the instant invention.
  • the effective daily amount ranges mentioned hereinabove are therefore only guidelines and are not intended to limit the scope or use of the invention to any extent.
  • the daily dosage regimen for an adult human patient may be, for example, an oral dose of between 0.01 mg and 3000 mg of each active ingredient, preferably between 1 mg and 700 mg, e.g. 5 to 200 mg.
  • the dosage may be a single one or a series of two or more given in the course of one or more days, as is needed by the subject.
  • the compounds will be administered for a period of continuous therapy, for example for a week or more, or for months or years.
  • human dosages for compounds have been established for at least some condition, those same dosages may be used, or dosages that are between about 0.1 % and 500%, more preferably between about 25% and 250% of the established human dosage.
  • a suitable human dosage can be inferred from ED50 or I D50 values, or other appropriate values derived from in vitro or in vivo studies, as qualified by toxicity studies and efficacy studies in animals.
  • dosages may be calculated as the free base.
  • dosages may be calculated as the free base.
  • Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the modulating effects, or minimal effective concentration (MEC).
  • MEC minimal effective concentration
  • the MEC will vary for each compound but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations. Dosage intervals can also be determined using MEC value.
  • Compositions should be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.
  • a compound of formula (V), where PG is benzyl is prepared from D-ribofuranose in two steps.
  • D-ribofuranose is methylated employing an acid such as H2SO4, in MeOH.
  • protection with a suitable protecting group such as benzyl employing conditions known to one skilled in the art, provides a compound of formula (V).
  • Removal of the methyl group in a compound of formula (V) is accomplished using an acid such a TFA, and the like, in water, for a period of 10-15 h, to provide a compound of formula (VI).
  • a compound of formula (VI), where PG is benzyl, is acetylated and subsequently treated with TMSCN/BF30Et2 to give a ribofuranosyl cyanide compound of formula (VII) and its epimer.
  • a compound of formula (VI) is also oxidized, employing oxidation conditions such as PCC, and the like, in a suitable solvent such as DCM, to provide a compound of formula (VIII).
  • a ribolactone compound of formula (VIII) is reacted with 3-((4-methoxybenzyl)oxy)isothiazole, in the presence of a base such as LDA, and the like, in a suitable solvent such as Et20, and the like, at -78 °C, to provide a compound of formula (X), where HET is isothiazole, and R a is -OPMB.
  • a ribofuranosyl thioamide compound of formula (XII) is cyclocondensed in a Hantzsch reaction, with ethyl ethyl 3-bromo-2-oxo-propanoate, in a suitable solvent such as EtOH, t-BuOH, and the like, employing conventional or microwave heating, to provide a thiazole compound of formula (XIII), where R b is C02Et, and PG is benzyl.
  • a thiadiazole compound of formula (XIII), where R b is C02Et, and PG is benzyl is synthesized in two steps from a ribofuranosyl thioamide compound of formula (XII), where PG is benzyl.
  • a ribofuranosyl thioamide compound of formula (XII) is reacted with commercially available or synthetically accessible ethyl 2- (dimethylamino)-2,2-diethoxyacetate (Intermediate 1 ), in a solvent such as ACN, and the like, employing microwave or conventional heating.
  • cyclization of the substituted carbothioamide in the presence of aminooxysulfonic acid (HAOS), a base such as pyridine, in a solvent such as EtOH, and the like, at a temperature of about 55 °C provides a thiadiazole compound of formula (XIII), where R b is C02Et, and PG is benzyl.
  • a compound of formula (VII), where PG is benzyl is reacted with sodium methoxide, subsequent hydrolysis of the resulting imidate in situ with HCI provides a methyl ester compound of formula (XIV).
  • a compound of formula (XV) is prepared in two steps from a compound of formula (XIV). In a first step, reaction with hydrazine, to provide the hydrazide intermediate, which in turn was acetylated with methyl 2-chloro-2-oxoacetate affords the substituted hydrazide of formula (XV).
  • oxidation of the vinyl R b moiety is achieved employing a catalyst such as Os04, an oxidant such as NMO, in a suitable solvent such as THF, acetone, water, or a mixture thereof, to provide a diol compound where R b is CH(OH)CH20H.
  • Oxidation of an aldehyde compound employing conditions known to one skilled in the art, provides a compound where R b is -CO2H.
  • the amine is ammonia
  • the dehydrating agent is HATU.
  • a compound of formula (XI), where R a is -SMe is oxidized employing conditions known to one skilled in the art.
  • reaction of a compound of formula (XI), where R a is -SMe with an oxidizing agent such as meta- chloroperoxybenzoic acid (mCPBA), in a suitable solvent such as DMC, at a
  • a compound of formula (XVII), where R b is CN, and R° is H is hydrolyzed to a compound of formula (XVII) where R b is -CO2H, under basic conditions.
  • reaction of a compound of formula (XVII), where R b is CN, and R° is H is hydrolyzed to a compound of formula (XVII) where R b is -CO2H, with a base such as KOH in a suitable solvent mixture such as MeOH, H2O, and THF, at a temperature of about 90 °C, for a period of 18 - 24 h, to provide a compound of formula (XVII) where R b is -CO2H.
  • Esterification of a compound of formula (XVII) where R b is -CO2H, is achieved using an alcohol such as 2-methylpropan-2-ol, and the like, DMAP, DCC, in a solvent such as DCM, to provide a compound of formula (XVII) where R b is -C02Ci-4alkyl.
  • a compound of formula (XIII) or (XVII), where R b is -C02Ci-4alkyl is reacted with a base such as LDA, at a temperature of about -78 °C, and a trialkyl tin reagent such as trimethyltin chloride, tributylchlorostannane, and the like, to provide a compound of formula (XVII) where R b is -C02Ci-4alkyl, and R° is Sn(Ci-4alkyl)3.
  • a compound of formula (XVII) where R b is - C02Ci-4alkyl, and R° is Sn(Ci-4alkyl)3, is fluorinated in a silver-mediated fluorination reaction.
  • a compound of formula (XVII) where R b is -C02Ci-4alkyl, and R° is Sn(Ci-4alkyl)3, is reacted with a suitable silver reagent such as Ag20, AgOTf, or a mixture therof, a fluorinating agent such as Selectfluor®, a base such as NaOH, K2CO3, NaHC03, and the like, in a suitable solvent such as acetone, EtOAc, and the like, at a temperature of about 65 °C, to provide a compound of formula (XVII), where R b is - C02Ci-4alkyl, and R° is F.
  • a suitable silver reagent such as Ag20, AgOTf, or a mixture therof
  • a fluorinating agent such as Selectfluor®
  • a base such as NaOH, K2CO3, NaHC03
  • a suitable solvent such as acetone, EtOAc, and the like
  • a compound of Formula (I) is acylated to provide a compound of Formula (II).
  • the secondary hydroxyl groups of a compound of Formula (I) are protected as oxomethylene-tethered, by treatment with trimethyl orthoformate and a catalytic amount of p-toluenesulfonic acid monohydrate.
  • a protected as oxomethylene-tethered compound is alternately reacted with Boc-valine, and the like, and DCC, in a suitable solvent such as DMF.
  • Compounds of Formula (I) may be converted to their corresponding salts using methods known to one of ordinary skill in the art.
  • an amine of Formula (I) is treated with trifluoroacetic acid, HCI, or citric acid in a solvent such as Et20, CH2CI2, THF, MeOH, chloroform, or isopropanol to provide the corresponding salt form.
  • trifluoroacetic acid or formic acid salts are obtained as a result of reverse phase HPLC purification conditions.
  • Cyrstalline forms of pharmaceutically acceptable salts of compounds of Formula (I) may be obtained in crystalline form by
  • the compounds according to this invention may accordingly exist as enantiomers. Where the compounds possess two or more chiral centers, they may additionally exist as diastereomers. It is to be understood that all such isomers and mixtures thereof are encompassed within the scope of the present invention.
  • Compounds prepared according to the schemes described above may be obtained as single forms, such as single enantiomers, by form-specific synthesis, or by resolution. Compounds prepared according to the schemes above may alternately be obtained as mixtures of various forms, such as racemic (1 : 1 ) or non-racemic (not 1 : 1 ) mixtures. Where racemic and non-racemic mixtures of enantiomers are obtained, single enantiomers may be isolated using conventional separation methods known to one of ordinary skill in the art, such as chiral chromatography, recrystallization, diastereomeric salt formation, derivatization into diastereomeric adducts, biotransformation, or enzymatic transformation. Where regioisomeric or diastereomeric mixtures are obtained, as applicable, single isomers may be separated using conventional methods such as chromatography or crystallization.
  • reaction mixtures were magnetically stirred at room temperature (rt) under a nitrogen atmosphere. Where solutions were“dried,” they were generally dried over a drying agent such as Na2S04 or MgS04. Where mixtures, solutions, and extracts were“concentrated”, they were concentrated on a rotary evaporator under reduced pressure. Reactions under microwave irradiation conditions were carried out in a Biotage Initiator.
  • Mass spectra were obtained on an Agilent series 1 100 MSD using electrospray ionization (ESI) in positive mode. Mass spectra of NTPs were obtained in negative mode. Calculated (calcd.) mass corresponds to the exact mass.
  • NMR Nuclear magnetic resonance
  • Step A Ethyl 2-(dimethylamino)-2-oxoacetate.
  • ethyl 2-chloro-2- oxoacetate 100.00 g, 732.45 mmol, 81.95 ml_
  • DCM 2.0 L
  • Et3N 133.4 g, 1.32 mol, 182.75 ml_
  • N-methylmethanamine hydrochloride 107.5 g
  • Step B Ethyl 2-(dimethylamino)-2.2-diethoxyacetate
  • Ethyl 2-(dimethylamino)-2- oxoacetate (90 g, 620 mmol, 1.00 eg.) was treated with triethyloxonium
  • Step A (2R,3S,4R)-2-(Hydroxymethyl)-5-methoxytetrahydrofuran-3,4-diol.
  • 3R,4S,5R -5-(hydroxymethyl)tetrahydrofuran-2,3,4-triol
  • H2SO4 (2.40 g, 23.98 mmol, 1.30 ml_, 98% purity).
  • the reaction mixture was stirred at 25 °C for 12 hours.
  • the reaction was set up for two batches.
  • the reaction mixture was diluted with MeOH (200 ml_), quenched with Na2C03 solid and filtered.
  • the filtrate was concentrated in vacuum. Purification (FCC, S1O2, DCM/MeOH from 25/1 to 5/1 ) afforded the title compound (40 g, 243.67 mmol, 91.45% yield) as colorless oil.
  • Step B (2R,3R,4R)-3,4-Bis(benzyloxy)-2-((benzyloxy)methyl)-5- methoxytetrahydrofuran.
  • 2R,3S,4R -2-(hydroxymethyl)-5- methoxytetrahydrofuran-3,4-diol (20.00 g, 121.83 mmol) in DMF (200.00 ml_) was added NaH (17.06 g, 426.41 mmol, 60% purity) at 0 °C.
  • the reaction mixture was stirred at 0 °C for 1 hour.
  • Step C (3R,4R,5R)-3,4-Bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-ol.
  • Step A (3R,4R,5R)-3,4-Bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-yl acetate.
  • TMSCN trimethylsilyl cyanide
  • Step A (2R,3R,4R,5R)-3,4-Bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2- carbothioamide.
  • Step C Ethyl 2-((2R,3R,4R,5R)-3,4-bis(benzyloxy)-5-
  • Step A (2R,3R,4R,5R)-3,4-Bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2- carboxylic acid.
  • Step B (2R.3R.4R.5R)-3.4-Bis(benzyloxy)-5-((benzyloxy)methyl)-N-methoxy-N- methyltetrahvdrofuran-2-carboxamide.
  • Step D 1 -((2R,3R,4R,5R)-3,4-Bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2- yl)-2-bromoethan-1 -one.
  • Step A (2R,3R,4R,5R)-3,4-Bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2- carbothioamide.
  • (2S,3S,4R,5R)-3,4-bis(benzyloxy)-5- ((benzyloxy)methyl)tetrahydrofuran-2-carbonitrile (Intermediate 4, 10.00 g, 23.28 mmol) in EtOH (300 ml_) and Et3N (50.00 ml_) was bubbling with hteS (15 PSI), and stirred at 25 °C for 1.5 h.
  • the reaction mixture was concentrated at low pressure.
  • Step B Ethyl (Z)-2-(((2R.3R.4R 5R)-3.4-bis(benzyloxy)-5-
  • Step D 5-((2R,3R,4R,5R)-3,4-Bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2- yl)-1 ,2,4-thiadiazole-3-carboxamide.
  • EtOH 5.00 ml_
  • Step E 5-((2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1 ,2,4- thiadiazole-3-carboxamide.
  • To a solution of 5-((2R,3R,4R,5R)-3,4-bis(benzyloxy)-5- ((benzyloxy)methyl)tetrahydrofuran-2-yl)-1 ,2,4-thiadiazole-3-carboxamide (405.00 mg, 763 mGhoI) in DCM (2.00 mL) was added BCb (1 M, 5.87 mL) at -78 °C. The mixture was stirred at 0 °C for 2 h.
  • Example 2 5-((2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2- yl)isothiazole-3-carboxamide.
  • Step A (3R,4R,5R)-3,4-Bis(benzyloxy)-5-((benzyloxy)methyl)-2-(3-((4- methoxybenzyl)oxy)isothiazol-5-yl)tetrahydrofuran-2-ol.
  • LDA 2 M, 2.87 ml_
  • Et20 5.00 ml_
  • Step B 5-((2R,3R,4R,5R)-3,4-Bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2- yl)isothiazol-3-ol.
  • Step C 5-((2R,3R,4R,5R)-3,4-Bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2- yl)isothiazol-3-yl trifluoromethanesulfonate.
  • Step D 5-((2R,3R,4R,5R)-3,4-Bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2- yl)isothiazole-3-carbonitrile.
  • Step E 5-((2R,3R,4R,5R)-3,4-Bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2- yl)isothiazole-3-carboxamide.
  • Step F 5-((2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2- yl)isothiazole-3-carboxamide.
  • To a solution of 5-((2R,3R,4R,5R)-3,4-bis(benzyloxy)-5- ((benzyloxy)methyl)tetrahydrofuran-2-yl)isothiazole-3-carboxamide (1 10.0 mg, 207.3 mP ⁇ qI) in DCM (2.00 ml_) was added BCb (1 M, 2.07 ml_) at -78°C under N2. The mixture was stirred at 0 °C for 2 h.
  • Example 3 2-((2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5- fluorothiazole-4-carboxamide.
  • Method B Ethyl 2-((2R,3R.4R.5R)-3.4-bis(benzyloxy)-5- ((benzyloxy)methyl)tetrahvdrofuran-2-yl)-5-fluorothiazole-4-carboxylate.
  • Step B Ethyl 2-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5- fluorothiazole-4-carboxylate.
  • ethyl 2-((2R,3R,4R,5R)-3,4-bis(benzyloxy)- 5-((benzyloxy)methyl)tetrahydrofuran-2-yl)-5-fluorothiazole-4-carboxylate 400.00 mg, 692.45 mGhoI) in DCM (7.00 ml_) was added a solution of BC (1 M, 6.92 ml_) at -78 °C.
  • Step C 2-((2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5- fluorothiazole-4-carboxamide.
  • Ethyl 2-((2R,3R,4S,5R)-3,4-dihydroxy-5- (hydroxymethyl)tetrahydrofuran-2-yl)-5-fluorothiazole-4-carboxylate (1 15.00 mg, 374.24 mGhoI) was treated with NH3 ⁇ 2O (2.50 mL, 25-28%). The reaction mixture was stirred at 18 °C for 15 minutes. The reaction mixture was concentrated under reduced pressure.
  • Example 4 ((2R 3S.4R.5R)-5-(3-Carbamoyl-1 2.4-thiadiazol-5-vn-3.4- dihvdroxvtetrahvdrofuran-2-vl)methvl isobutvrate.
  • Step A 5-((3aR,4R,6R,6aR)-6-(Hydroxymethyl)-2-methoxytetrahydrofuro[3,4- dlH .31dioxol-4-yl)-1.2.4-thiadiazele-3-carbexamide.
  • Step B ((3aR 4R.6R.6aR)-6-(3-Carbamoyl-1 2.4-thiadiazol-5-vn-2- methoxytetrahvdrofuro[3,4-d1[1 ,31dioxol-4-yl)methyl isobutyrate.
  • Step C ((2R,3S,4R,5R)-5-(3-Carbamoyl-1 ,2.4-thiadiazol-5-yl)-3,4- dihydroxytetrahydrofuran-2-yl)methyl isobutyrate. ((3aR,4R,6R,6aR)-6-(3-Carbamoyl-
  • MeOH/CH2Cl2 solvent system (2-10% gradient) to yield 20 mg of 2’, 3’- methoxymethylene derivative.
  • Step B The intermediate from Step A is reacted with triethylammonium
  • Example 5 (27 mg, 40% for 2 steps) in the same manner as described for Example 4.
  • MS: m/z 616 (M+1 ) + .
  • Example 5 A solution of Example 5 (27 mg, 0.044 mmol) and 80% aq. AcOH (2 ml_) was stirred at r. t. for 3 h. The mixture was then concentrated. Thus obtained residue was co evaporated several times with toluene followed by methanol containing few drops of Et3N. The evaporated residue was purified on silica gel column with MeOH/ChhC solvent system (3-12% gradient) to yield Example 6 (20 mg, 80%).
  • Step A 5-((3aR,4R,6R,6aR)-6-(Hydroxymethyl)-2-methoxytetrahydrofuro[3,4- dlH .31dioxol-4-yl)-1.2.4-thiadiazole-3-carboxamide.
  • Methyl orthoformate was added (210 mI_, 2 mmol) followed by TsOH (76 mg, 0.4 mmol). The mixture was left overnight at ambient temperature. Methanol (5 ml_) and Et3N (0.5 ml_) was added and left for 30 min. at ambient temperature. The reaction mixture was concentrated under reduced pressure. Purification (FCC, S1O2, methanol in DCM from 2% to 10%) afforded 40 mg of the title compound.
  • Step B ((3aR,4R,6R,6aR)-6-(3-Carbamoyl-1 ,2,4-thiadiazol-5-yl)-2- methoxytetrahydrofuror3,4-diri ,31dioxol-4-yl)methyl valinate.
  • Step C ((2R,3S,4R,5R)-5-(3-Carbamoyl-1 ,2,4-thiadiazol-5-yl)-3,4- dihydroxytetrahydrofuran-2-yl)methyl valinate.
  • a solution of ((3aR,4R,6R,6aR)-6-(3- carbamoyl-1 ,2,4-thiadiazol-5-yl)-2-methoxytetrahydrofuro[3,4-d][1 ,3]dioxol-4-yl)methyl valinate was treated with 1 N HCI/dioxane-DCM 1 : 1 (v/v) solution for 40 min., and concentrated at low pressure.
  • reaction was diluted with water (10 ml_) and loaded on the column HiLoad 16/10 with Q Sepharose High Performance. Separation was done in a linear gradient of NaCI from 0 to 1 N in 50mM TRIS-buffer (pH7.5). Triphosphate was eluted at 75-80%B. Corresponding fractions were concentrated. Desalting was achieved by RP HPLC on Synergy 4 micron Hydro-RP column (Phenominex). A linear gradient of methanol from 0 to 30% in 50mM
  • triethylammonium acetate buffer pH 7.5 was used for elution. The corresponding fractions were combined, concentrated and lyophilized 3 times to remove excess of buffer.
  • Example 9 ((2R.3S.4R.5R)-5-(4-Carbamoyl-5-fluorothiazol-2-yl)-3.4- dihydroxytetrahydrofuran-2-yl)methyl tetrahydroqen triphosphate.
  • Example 10 ((2R 3S 4R.5R)-5-(3-Carbamoyl-1 2 4-thiadiazol-5-yl)-3.4- dihydroxytetrahydrofuran-2-yl)methyl tetrahydroqen triphosphate.
  • Example 1 1 ((2R,3S,4R,5R)-5-(3-Carbamoylisothiazol-5-yl)-3,4- dihydroxytetrahydrofuran-2-yl)methyl tetrahydroqen triphosphate.
  • Example 12 ((2R,3S,4R,5R)-5-(3-Carbamoyl-4-fluoroisothiazol-5-yl)-3,4- dihydroxytetrahydrofuran-2-yl)methyl tetrahydroqen triphosphate.
  • Step A 5-((2R,3R,4R,5R)-3,4-Bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2- yl)isothiazole-3-carbonitrile.
  • the title compound is Example 3, product from Step D.
  • Step B 5-((2R,3R,4R,5R)-3,4-Bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2- yl)isothiazole-3-carboxylic acid.
  • Step C tert-Butyl 5-((2R,3R,4R,5R)-3,4-bis(benzyloxv)-5-
  • reaction was set up for two batches.
  • the reaction mixture was filtered and the filter was concentrated under reduced pressure.
  • Step D tert-Butyl 5-((2R,3R,4R,5R)-3,4-bis(benzyloxy)-5-
  • Step E tert-Butyl 5-((2R,3R,4R,5R)-3,4-bis(benzyloxv)-5-
  • Step G 5-((2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4- fluoroisothiazole-3-carboxamide.
  • 5-((2R,3R,4R,5R)-3,4-bis(benzyloxy)- 5-((benzyloxy)methyl)tetrahydrofuran-2-yl)-4-fluoroisothiazole-3-carboxamide 130 mg, 236.96 mGhoI) in DCM (1 ml_) was added BCI3 (1 M, 2.37 ml_, 10 eq.) at -78 °C under N2.
  • Step H ((2R,3S,4R,5R)-5-(3-Carbamoyl-4-fluoroisothiazol-5-yl)-3,4- dihydroxytetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate.
  • Example 13 ((2R,3S,4R,5S)-5-(2-Carbamoyloxazol-4-yl)-3,4-dihydroxytetrahydrofuran-
  • Step A 4-((2S,3S,4R,5R)-3,4-Bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2- yl)-2-vinyloxazole.
  • 1 -((2R,3R,4R,5R)-3,4-bis(benzyloxy)-5- ((benzyloxy)methyl)tetrahydrofuran-2-yl)-2-bromoethan-1 -one (Intermediate 8, 3 g, 5.71 mmol) in EA (30 ml_) was added s triflate (1.91 g, 7.42 mmol) and acrylamide (527.58 mg, 7.42 mmol). The mixture was stirred at 70 °C for 12 h. The reaction mixture was cooled down and filtered, and the filtrate was concentrated at low pressure. The residue was purified by column chromatography (FCC, S1O2, Petroleum ether/Ethyl
  • Step B 1 -(4-((2S.3S.4R.5R)-3.4-Bis(benzyloxy)-5-((benzyloxy)methyl)tetrahvdrofuran-
  • Step C 4-((2S,3S,4R,5R)-3,4-Bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2- yl)oxazole-2-carbaldehyde.
  • Step D 4-((2S,3S,4R,5R)-3,4-Bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2- yl)oxazole-2-carboxylic acid.
  • reaction mixture was diluted with water (20 ml_).
  • the reaction mixture was extracted by EA (10 mL x 2) and the organic layer was washed with brine (10 mL), dried over anhydrous Na2S04, and concentrated at low pressure.
  • LCMS: ESI -MS: m/z 538.1 [M + Na] + .
  • Step E 4-((2S,3S,4R,5R)-3,4-Bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2- yl)oxazole-2-carboxamide.
  • Step F 4-((2S.3R.4S.5R)-3.4-Dihvdroxy-5-(hvdroxymethyl)tetrahvdrofuran-2-yl)oxazole- 2-carboxamide.
  • 4-((2S,3S,4R,5R)-3,4-bis(benzyloxy)-5- ((benzyloxy)methyl)tetrahydrofuran-2-yl)oxazole-2-carboxamide 150 mg, 271.10 mGhoI
  • BCb (1 M, 2.71 ml_, 10 eg in DCM ) at -78 °C and stirred at 0 °C for 2 h.
  • Step G ((2R,3S,4R,5S)-5-(2-Carbamoyloxazol-4-yl)-3,4-dihydroxytetrahydrofuran-2- yl)methyl tetrahydrogen triphosphate.
  • Human lung carcinoma A549 cells (ATCC, Manassas, VA) were plated at a density of 5 x 10 4 cells/ml (5 x 10 3 cells/well) in maintenance media (Ham’s F12 media supplemented with 10% FBS, 1 % penicillin/streptomycin, 1 % HEPES, 1 % Glutamine and 1 % non-essential amino acids (all Mediatech, Manassas, VA) in 96-well plates.
  • maintenance media Ham’s F12 media supplemented with 10% FBS, 1 % penicillin/streptomycin, 1 % HEPES, 1 % Glutamine and 1 % non-essential amino acids (all Mediatech, Manassas, VA) in 96-well plates.
  • Luminescence was measured on a Victor X3 multi-label plate reader.
  • Madin-Darby canine kidney epithelial cells (MDCK, ATCC), were plated at a density of 7.5 x 10 4 cells/ml (7.5 x 10 3 cells/well) in maintenance media (DMEM with same supplements as above) in 96-well plates. After 24 hours, serially diluted compounds in assay media (MEM supplemented with 0.3FBS, 1 %
  • the cell culture supernatant was aspirated off and 50 pL 25 pM 2’-(4- Methylumbelliferyl)-a-D-N-acetylneuraminic acid (MUNANA, Sigma-Aldrich) dissolved in 33 mM MES, pH 6.5 (Emerald Biosystems, Bainbridge Island, WA) was added to the cells. After incubation for 45 min at 30°C, reactions were stopped by addition of 150 pi stop solution (100 mM glycine, pH 10.5, 25% ethanol, all Sigma-Aldrich). Fluorescence was measured with excitation and emission filters of 355 and 460 nm, respectively, on a Victor X3 multi-label plate reader (Perkin Elmer, Waltham, MA).
  • Cytotoxicity of uninfected parallel cultures was determined by addition of 100 pL CellTiter-Glo®reagent (Promega, Madison, Wl), and incubation for 10 min at room temperature. Luminescence was measured on a Victor X3 multi-label plate reader.
  • n.d. means not determined
  • the nucleotide incorporation activity of IAV PA/PB1/PB2 complex is measured as an incorporation of tritiated UMP into acid-insoluble RNA products.
  • the reactions contain 30 nM recombinant enzyme, 100 nM IAV mini-genome RNA, 0.5 mM 5’vRNA, 100 pM ATP, 100 pM GTP, 100 pM CTP, 0.5 pM tritiated UTP, 40 mM Tris-HCI (pH 7.4), 0.4U/DL RNaseln, 0.2 mg/ml_
  • RNA is precipitated with 10% TCA, and acid-insoluble RNA products are filtered on a size exclusion 96-well plate. After washing of the plate, scintillation liquid is added and radiolabeled RNA products are detected according to standard procedures with a Trilux Topcount scintillation counter.
  • the compound concentration at which the enzyme-catalyzed product formation rate is reduced by 50% is calculated by non linear regression data fitting to a sigmoidal dose-response equation.
  • the RSV subgenomic replicon 395 Hela was licensed from Apath (Brooklyn, NY) and was originally developed by Dr. Mark Meeples of Center for Vaccines & Immunity, the Research Institute at National Children's Hospital in Columbus, Ohio [2]
  • three glycoprotein genes, those for SH, G, and F, from a full-length recombinant GFP-expressing (rg)RSV antigenomic cDNA were deleted.
  • a blasticidin S deaminase ( bsd) gene was inserted.
  • the RSV replicon was established in Hela cells.
  • the 395 Hela cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 4500 mg/L
  • DMEM Modified Eagle Medium
  • the medium was further supplemented with 10% (v/v) fetal bovine serum (FBS) (Mediatech, Cat. #35-010-CV), 1 % (v/v) penicillin/streptomycin (Mediatech, Cat. #30-002-CI), and 10 pg/mL of Blasticidin (BSD) (Invivogen, Cat. # ant-bl-1 ).
  • FBS fetal bovine serum
  • BSD Blasticidin
  • ICso 50% inhibitory concentration
  • IC90 90% inhibitory concentration
  • CC50 50% cytotoxic concentration
  • the compounds to be tested were solubilized in 100% DMSO to 100 x the desired final testing concentration. Each compound was serially diluted (1 :3) up to 9 distinct concentrations. Compounds in 100% DMSO were reduced to 10% (v/v) DMSO by diluting 1 : 10 in cell culture media. A 10 pL sample of the compounds diluted to 10% (v/v) DMSO with cell culture media was used to treat the RSV replicon cells in 96-well format. The final DMSO concentration was 1 %
  • the Renilla Luciferase Assay System (Promega, Cat. # E2820) was used to measure anti-RSV replicon activity. Assay plates were set up as stated above (see
  • Luminescence was recorded using a Perkin Elmer multilabel counter Victor3V.
  • IC50 the concentration of the drug required for reducing RSV replicon RNA by 50% in relation to the untreated cell control value, was calculated from the plot of percentage reductions of the optical density (OD) value against the drug concentrations using the Microsoft Excel forecast function.
  • Luminescent Cell Viability Assay is a homogeneous method to determine the number of viable cells in culture based on quantitation of the ATP present, which signals the presence of metaboiica!iy active ceils. Assay plates were set up in the same format as in the replicon assay (see Section 4.4). CellTiter-Glo reagent (100 pL) was added to each well and incubated at room temperature for 8 minutes. Luminescence was recorded using a Perkin Elmer multilabel counter Victor3V. The CCso, the concentration of the drug required for reducing viable cells by 50% in relation to the untreated cell control value, was calculated from the plot of percentage reductions of the

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